A total of 60 female adult Sprague-Dawley rats (weighing 200–250 g) were obtained from the Animal Centre of Sun Yat-sen University (Guangzhou, China). The use and care of animals, in addition to the experimental and surgical procedures, were reviewed and approved by the Institutional Animal Care and Use Committee of Sun Yat-Sen University and the Ethics Committee of Sun Yat-Sen University. The animals were housed under a 14 h:10 h light-dark cycle at room temperature between 18 and 26°C and 60–70% humidity. Food and water were available ad libitum. The rats were randomly divided into 10 groups (6 rats/group): Normal saline (NS), SEV, apocynin (AP), sham, IIR, IIR + compound 48/80 (CP), SEV + IIR, SEV + IIR + CP, AP + IIR and AP + IIR + CP). The treatments administered to these groups was as follows: NS group, 1 ml NS only; SEV group, 2.3% SEV inhalation only; iii) AP group, 2.5 mg/kg AP only; sham, administration of 1 ml NS each day for 3 days prior to surgical isolation of the superior mesenteric artery (SMA) without occlusion; IIR, administration of 1 ml NS each day for 3 days prior to IIR, established by occluding the SMA for 75 min, followed by 2 h reperfusion; IIR + CP, IIR with administration of CP (0.75 mg/kg) via the caudal vein 5 min prior to reperfusion; vii) SEV + IIR, IIR following exposure to 2.3% SEV each day for 3 days; SEV + IIR + CP, IIR + CP following exposure to 2.3% SEV each day for 3 days; AP + IIR, IIR following pretreatment with AP each day for 3 days; and AP + IIR + CP, IIR + CP following pretreatment with AP each day for 3 days. In the NS, AP and SEV groups, the animals did not undergo surgery, but received saline (1 ml; KeyGen Biotech. Co., Ltd., Nanjing, China) or AP (2.5 mg/kg; Sigma-Aldrich, St. Louis, MO, USA) via intraperitoneal injection, or 2.3% SEV (Maruishi Pharmaceutical Co., Ltd., Osaka, Japan) by inhalation. In the sham group, the SMA was isolated, but not clamped; in the IIR group, all rats received SMA separation and clipping for 75 min, followed by 2 h reperfusion. For the groups treated with NS, AP or SEV, the rats were injected with NS (1 ml) or AP (2.5 mg/kg), or administered with 2.3% SEV via inhalation for three consecutive days prior to surgery. For the groups treated with CP, 0.75 mg/kg CP (Sigma-Aldrich) was injected via the caudal vein 5 min prior to reperfusion. The rats in the remaining IIR groups received the same volume of normal saline (1 ml). The experimental procedure used in the present study is presented in Fig. 1. A heating lamp was used to maintain the body temperatures of the rats. The optimal doses of SEV (2.3%), AP (2.5 mg/kg) and CP (0.75 mg/kg) were adjusted, in accordance with those previously described (24–26) with modifications.

The rats were sacrificed through overdose of pentobarbital (70 mg/kg; intraperitoneal injection; Sigma-Aldrich) 2 h after reperfusion. Blood samples (2 ml) were obtained from the abdominal aorta and centrifuged (Centrifuge 5804; Eppendorf, Hamburg, Germany) at 1,699 × g for 15 min at 4°C, and the resultant plasma samples were stored at -80°C until analysis. A thoracotomy was immediately performed and the right upper lung was removed, fixed in 10% formaldehyde (Sigma-Aldrich) and embedded in paraffin (Leica Biosystems, Nussloch, Germany) for sectioning. The middle lobe of the right lung was removed and used to measure the wet/dry (w/d) weight ratio. The inferior lobes of the right and left lungs were removed and preserved in liquid nitrogen (Guangzhou Pearl River Industrial Gases Co. Ltd., Guangzhou, China) for further analysis of oxidative stress, mast cell activation and inflammatory indicators.

The middle lobes of the right lungs were weighed (weight_wet) immediately using a precision balance [Mettler-Toledo (Schweiz) GmbH, Greifensee, Switzerland], and re-weighed (weight_dry) following incubation at 95°C in an oven (876–1 Vacuum Drying Oven; Nantong Science Instrument Factory, Nantong, China) for 24 h. The w/d ratio was calculated as follows: w/d = weight_wet / weight_dry.

The lung tissues embedded in paraffin were dissected into 4-μm microsections (ZQP-86; Zhejiang Xiangshan Scientific Precision Instrument Factory, Xiangshan, China), which were stained and observed under a light microscope (Eclipse E200; Nikon, Tokyo, Japan). Hematoxylin and eosin (Beyotime Institute Biotechnology, Shanghai, China) staining was used to assess pathological injury, while staining with toluidine blue (Beijing Leagene Biotech Co., Ltd., Beijing, China) was used to count the number of mast cells. The degree of lung injury was assessed using a scoring system, described by Hofbauer et al (27). According to this scoring system, edema of the alveoli and mesenchyme, intra-alveolar inflammatory cell infiltrates, alveolar hemorrhage and atelectasis were graded on a scale between 0 and 4. The grades were as follows: 0, normal, <15% of space occupied by tissue and >85% occupied by alveolar space; 1, 15%–25% of space occupied by tissue and 75%-85% occupied by alveolar space; 2, 25%–50% occupied by tissue and 50%–75% occupied by alveolar space; 3, 50%–75% occupied by tissue and 25%–50% occupied by alveolar space; and 4, 75%–100% occupied by tissue and 0%–25% occupied by alveolar space. For the sections stained with toluidine blue, cells containing blue-purple granules in the cytoplasm were considered to be mast cells.

β-hexosaminidase is one of the specific enzymes synthesized by mast cells, and mast cell activation is accompanied by the release of histamine and β-hexosaminidase. While histamine is metabolized rapidly, β-hexosaminidase is metabolized less readily and can be used as an index of mast cell activation (28). In the present study, the lung tissues were homogenized with cold normal saline and the homogenates were centrifuged at 1,699 × g for 15 min at 4°C. The supernatants were then transferred into fresh tubes for detection. The activities of β-hexosaminidse in the lung tissue homogenates and blood samples were detected using a β-hexosaminidase kit, according to the manufacturer’s instructions (Nanjing KeyGen Biotech. Co., Ltd.).

The levels of MDA and H₂O₂ in the homogenates of the lung tissues were measured using the MDA Detection kit and the H₂O₂ Detection kit, according to the manufacturer’s instructions (KeyGen Biotech. Co., Ltd.).

The concentration of IL-6 is an independent marker for the inflammatory response. MPO is an enzyme induced by activated neutrophils and is considered an indicator of neutrophil infiltration (29,30). The activities of IL-6 and MPO in the lung tissues were measured using the IL-6 Detection kit and the MPO Detection kit (KeyGen Biotech Co., Ltd.), according to the manufacturer’s instructions.

Prosurfactant protein C (proSP-C) is the precursor of surfactant protein C, which is exclusively produced in alveolar type II cells to prevent lung collapse (31). Trypatse is one of the characteristic markers of mast cell activation (32). The expression of NADPH oxidase reflects the level of oxidative stress, and p47^phox and gp91^phox are subunits of NADPH oxidase (33). Intercellular adhesion molecule-1 (ICAM-1, CD54) is an important early marker of immune activation and response (34). Western blot analyses were performed, as previously described (24). The primary antibodies used in the present study were as follows: Anti-ICAM-1 mouse monoclonal antibody (1:500; sc-8439; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), antitryptase rabbit polyclonal antibody (1:500; sc-32889; Santa Cruz Biotechnology, Inc.), anti-proSP-C polyclonal rabbit antibody (1:1000; AB3786; EMD Millipore, Billerica, MA, USA), anti-p47^phox polyclonal rabbit antibody (1:1,000; sc-14015; Santa Cruz Biotechnology, Inc.) and anti-gp91^phox polyclonal rabbit antibody (1:1,000; sc-20782; Santa Cruz Biotechnology, Inc.). The images were analyzed used ImageJ software, version 1.41 (National Institutes of Health, Bethesda, MA, USA).

All data are expressed as the mean ± standard deviation. Statistical analyses were performed using SPSS software, version 13.0 (SPSS Inc., Chicago, IL, USA). The statistical significance of differences between the groups were evaluated by one-way analysis of variance followed by Bonferroni’s post-hoc test for unpaired values. P<0.05 was considered to indicate a statistically significant difference.

As shown in Fig. 2A, the pulmonary structures were normal following treatments with NS, SEV and AP for 3 days. Treatment with SEV or AP did not significantly change the number of mast cells (Fig. 2B), expression of tryptase or the levels of β-hexosaminidase in either the plasma or the lung tissues (Fig. 2C). However, the expression levels of p47^phox and gp91^phox were downregulated in the lungs (P<0.05), inhibiting NAPDH enzyme activity (Fig. 2D).

As shown in Fig. 3A, the lung structures in the sham group were normal. IIR resulted in severe damage to the lungs, with collapse of the alveoli, interstitial edema, haemorrhage in the alveoli and mesenchyme, neutrophil infiltration and atelectasis. In addition, treatment with CP aggravated IIR-induced lung injury. Pretreatment with SEV and AP significantly prevented the lung damage induced by IIR and IIR + CP. The pathological injury score and w/d ratio in the lungs were in accordance with the pathological changes observed under the light microscope (Fig. 3B and C).

The expression levels of proSP-C were significantly reduced in the IIR group and were reduced further in the IIR + CP group compared with the sham group (Fig. 4). SEV and AP inhibited the downregulated expression of proSP-C induced by IIR and IIR + CP. These results suggested that IIR caused damage to type II alveolar epithelial cells, that mast cell degranulation exacerbated the damage, and that SEV and AP protected the type II alveolar epithelial cells from the injury induced by IIR and mast cell degranulation.

As shown in Fig. 5, IIR resulted in an increase in the number of mast cells and expression levels of tryptase in the lung tissues, and the levels of β-hexosaminidase in the lungs and plasma. CP aggravated these changes. Pretreatment with SEV and AP effectively alleviated the changes induced by IIR and IIR + CP.

NADPH oxidase is crucial for ROS generation, and p47^phox and gp91^phox are the predominant subunits of NADPH oxidase (35). As shown in Fig. 6, IIR increased the expression levels of p47^phox and gp91^phox in the lungs, which were further upregulated by CP. Pretreatment with SEV and AP significantly reversed this upregulation in the expression levels of p47^phox and gp91^phox. The changes observed in the levels of H₂O₂ and MDA were consistent with the changes observed in the expression of p47^phox and gp91^phox. These results suggested that SEV and AP alleviated the oxidative injury in the lungs, induced by IIR, by inhibiting the activity of NADPH oxidase.

As shown in Fig. 7, IIR increased the levels of IL-6, activity of MPO and the protein expression levels of ICAM-1 levels, and these were increased further by CP. Pretreatment with SEV and AP effectively reduced the levels of IL-6, activity of MPO and protein expression of ICAM-1, which was induced by IIR and IIR + CP. These results suggested that SEV and AP inhibited IIR-induced lung injury by inhibiting inflammatory responses.